The present disclosure relates to a laser scanning microscope system and a method of setting a laser-light intensity value in view of cell phototoxicity and fluorescence fading.
In recent years, laser microscopes such as confocal microscopes and multiphoton excitation microscopes are designed in various ways to control intensity values of emission from laser light sources, in order to reduce phototoxic damages to samples such as cells.
For example, according to the technology disclosed in Japanese Patent Application Laid-open No. 2012-212133, sectional images of a sample is obtained in order from the surface of the sample in the depth direction. A laser-light intensity value is controlled based on the fluorescence distribution of those sectional images. In other words, the laser-light intensity value is controlled based on the depth from the surface of the sample. As a result, according to this document, fluorescence images may not be affected by scattering and the like resulting from the thickness of the sample, and fluorescence images having the same brightness may be obtained.
Moreover, when a fluorescent-dyed sample is observed, a fluorescent material is excited and fading of fluorescence occurs as a result, which is problematic. For example, the technology disclosed in Japanese Patent Application Laid-open No. 2012-128354 deals with the fading problem. According to this technology, in order to deal with fluorescence fading, when a plurality of images are taken, the laser-light intensity value is changed from a high intensity value to a low intensity value, and the sensitivity of a photodetector is changed from high sensitivity to low sensitivity. A high laser-light intensity value and high sensitivity are selected before fluorescence is faded, and then an image is taken. As a result, an image of a low brightness area of a cell, i.e., a sample, may be taken sharply. According to this technology, a plurality of images having different fluorescence intensity values are taken, and those images are synthesized. As a result, a synthesized image having a large dynamic range may be obtained.